Molecular replacement applications

The rule of thumb for a successful application of molecular replacement is that the model should have a root-mean-square deviation (RMSD) on C-alpha coordinates 2.0-2.5 Angstroms with the target structure, corresponding to a sequence identity with the target of 25-35%. In practice, however, there are many more structures solved by MR in the PDB using models with sequence identity of 60% or higher than otherwise. 

A way to get round collecting and processing image sets for each new ligand tested was to use the phases from the original PTX experiment to process the diffraction amplitudes recorded for new drugs in the Zn sheets. As discussed above, this is not a new concept. The techniques molecular replacement (MR) and phase switching are often employed in X-ray crystal structure determination when actual phases aren t known [32], The application and results of these experiments are described herein. 

The first structure of human renin was obtained from prorenin produced by expression of its cDNA in transfected mammalian cells. Prorenin was cleaved in the laboratory to renin using the protease trypsin. Because the carbohydrates in renin are not required for bioactivity, oligosaccharides were removed enzymatically. This process facilitates crystallization in some cases and also removes the contribution of the heterogeneous sugar chains to the diffraction pattern. The structure was determined without the use of heavy-atom derivatives, by application of molecular replacement techniques based on the atomic coordinates of porcine pepsinogen as the model. The molecular dynamic method of refinement was used extensively to arrive at a 2.5 A resolution structure. However, some of the loop regions were not well resolved in this structure (Sielecki et al, 1989 Sail et al, 1990). 

It is possible, as shown by Rossmann and Blow (1962), to search for redundancies in Patterson space that correspond to the multiple copies of molecular transforms. Rossmann and Blow show, however, that the Patterson map does not need to be computed and used in any graphical sense, but that an equivalent search process can be carried out directly in diffraction or reciprocal space. Using such a search procedure, called a rotation function, they showed that noncrystallographic relationships, both proper and improper rotations, could be deduced in many cases directly from the X-ray intensity data alone, and in the complete absence of phase information. Translational relationships (only after rotations have been established) can also be deduced by a similar approach. Rotation functions and translation functions constitute what we call molecular replacement procedures. Ultimately the spatial relationships among multiple molecules in an asymmetric unit can be defined by their application. 

Molecular replacement (see Rossmann, 1972) has another very useful application, one that has for the most part superseded that described above for directly deducing phase information from noncrystallographic relationships. It derives from the tendency of macromolecules to fall into classes with shared structural motifs, in that they are homologous and often evolutionarily related. Macromolecules of similar function from different species often share structural features and are frequently almost identical. Viruses of the same or similar families do as well. Protein domains of common occurrence may serve as modules to be assembled in different combinations to make a variety of proteins having redundant structural features. 

As more macromolecular structures become known through X-ray crystallography, then this form of molecular replacement will see ever greater application. With sequence information to guide us, we may eventually be able to accurately and confidently predict what known model structure should be chosen to determine the approximate phases for any new but still unknown macromolecular crystal. 

The application of molecular replacement relies on similar model proteins. If there are no known proteins that are similar to the proteins we want to study, then in such cases, what we face are the de novo structures, and molecular replacement is not valid any more. Isomorphous replacement or anomalous scattering methods have to apply in order to solve the phase problems. 

This method can sometimes be used for determining the probable elemental composition of fragment ions. However, it is not as generally applicable and does not replace accurate mass measurement for determining molecular formulae and elemental compositions. 

This result must now, however, be replaced by one based upon the quantum theory. In this case a generally applicable expression cannot be obtained, and it is necessary to consider particular molecular models. W. Pauli, Jr.3 has treated the diatomic dipole, which is of interest to us, and his treatment forms the basis of this discussion. 

A more significant body of literature focuses on the use of protoplasts in understanding processes related to stress tolerance. The role of Ca in salt toleranee has been evaluated using maize root protoplasts. Exposure of the plasmalemma directly to external media revealed a non-specific replacement of Ca by salt. Sodium was found to replace Ca though this could be reversed by adding more Ca (Lynch, Cramer Lauchli, 1987). This approach assists in understanding the role of specific ion interaction in enhancing salt tolerance and is potentially applicable to studies on the molecular basis for ion specificity of plant membranes. 

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